Electronic Supplementary Material (ESI) for Environmental Science: Nano. This journal is The Royal Society of Chemistry 2017 Effects of titanium oxide nanoparticles on tetracycline accumulation and toxicity in Oryza sativa (L.) Chuanxin Ma,,, Hong Liu,,, *, Guangcai Chen Γ, Qing Zhao, Brian Eitzer, Zonghua Wang Ɨ, Wenjun Cai Ԓ, Lee A. Newman Ԓ, Jason C. White, Om Parkash Dhankher, and Baoshan Xing, * College of Resources and Environmental Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, United States Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States Γ Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang 311400, China Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China Ɨ State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China Ԓ Department of Environmental and Forest Biology, SUNY - College of Environmental Science and Forestry, Syracuse, New York 13210, United States Corresponding authors: * Baoshan Xing: bx@umass.edu; Phone: 413-545-5212; Fax: 413-577-0242; * Hong Liu: fjauliuhong@163.com; Phone: 0591-83765330; Fax: 0591-83776849 These authors contributed equally to this work. Numbers of Pages: 22 Numbers of Figures: 12 Numbers of Tables: 6 S1
S1. Adsorption isothermal experiment One and a half mg of TiO2 NPs were weighed into 8 ml glass tubes containing 8 ml 1/2x Hoagland s solution (ph 5.7) amended with different concentrations of TC (0, 0.5, 1.0, 2.5, 5, 7.5, 10.0, 12.5, 15, 20 mg/l). The glass tubes were covered with aluminum-foil lined caps and shaken end-to-end in a rotator at 25 ºC. The suspension was sampled at day 3 (as determined by the kinetic experiment) and filtered with 0.22 µm filter. The TC concentrations were determined using HPLC (Waters 1525) equipped with a UV-detector (Waters 2487) at 355 nm with a C18 column. The mobile phase was a mixture of acetonitrile and oxalic acid (15/8, v/v). The mobile phase was adjusted to ph of 2 and flow rate was 1 ml/min. The triplicate was set in the experiment. The adsorption amount was calculated as the followed equation. where q (mg/g) is the amount of TC adsorbed onto the NPs; C0 and C t is the initial and that at time t (mg/l) concentrations of TC, respectively (calculated based on the standard curve of TC); qc (mg/g) is the amount of TC in control without sorbents (TiO2 NPs); V (L) is the initial volume of the solution; W (g) is the weight of the NPs. S2. Analysis for protein content and antioxidant enzyme activities For protein content determination, plant fine powder was vigorously mixed with 10 mm Tris- HCl (ph 7.2) at a ratio of 1:10 (w/v). The mixture was centrifuged at 4000 rpm, 4 C for 20 min. One hundred μl of supernatant and 1900 μl of Bradford reagent were reacted in a 2 ml centrifuge tube for 15 min at ambient temperature. The absorbance of each sample was measured at 595 nm. Detailed information for antioxidant enzyme extraction buffer, reaction buffer, reaction time, as well as wavelength, are shown in Table S3. S2
S4. TiO2 NPs characterization as affected by TC The hydrodynamic diameter and zeta potential of TiO2 NPs in single analyte and co-exposure treatments were determined in 1/2x Hoagland s solution (Figure S5 and Table S2). In TiO2 NPs alone treatments, the hydrodynamic diameters decreased with increasing concentration of NPs in 1/2x Hoagland s solution. One of the possible explanations was that large aggregates might settle down before measurements. The surfaces of TiO2 NPs were all negatively charged. Interestingly, the addition of different concentrations of TC notably decreased the hydrodynamic diameter of TiO2 NPs regardless of the doses of TiO2 NPs. The values of zeta potential also suggested that the presence of TC caused positive charges on the surface of TiO2 NPs, which could be ascribed to that pka value of TC is positive (3.3 9.7) at 25 C. However, such alteration was only evident in 1000 and 2000 mg/l TiO2 NPs amended 1/2x Hoagland s solution. S5. The total protein contents in rice seedlings treated with TiO2 NPs TC As shown in Figure S11, the presence of TiO2 NPs and TC significantly altered the total protein contents in rice shoots and roots. For rice shoots, 2000 mg/l TiO2 NPs alone and 20 mg/l TC alone significantly increased the total protein content by approximately 30% relative to the control. In the co-exposure scenarios, elevation of the total protein content was evident as compared to 5 mg/l and 10 mg/l TC alone treatment. However, in the 20 mg/l TC treatment, the presence of TiO2 NPs did not further increase the protein content, which were all decreased by 18.1% relative to its TC alone treatment; these values were still significantly higher than the control. Similar to the shoots, the protein contents in rice roots were significantly increased upon exposure to TiO2 NPs alone or TC alone, regardless of the dose. It is worth mentioning that co- S3
exposure of TiO2 NPs and TC resulted in decreases of the root proteins as compared to the control or the single contaminant treatment. For example, in the 10 and 20 mg/l TC treatments with 2000 mg/l TiO2 NPs, the total protein levels were only 0.5-fold of the control, or 0.2- and 0.12-fold of the respective TC alone treatment. S4
A B A Figure S1. Phenotypic images of rice seedlings treated with different concentrations of TC in 1/2x Hoagland s solution. (A) and (B) represent images of whole seedlings and rice roots, respectively, in response to different concentrations of TC. S5
Figure S2. Seedling length and fresh biomass of different concentrations of TC treated rice. (A) shows root length and shoot height; (B) represents fresh biomass of rice shoots and roots. S6
A A B A Figure S3. Phenotypic images of rice seedlings treated with different concentrations of TiO2 NPs in 1/2x Hoagland s solution. (A) and (B) represent images of whole seedlings and rice roots, respectively, in response to different concentrations of TiO2 NPs. S7
Figure S4. Seedling length and fresh biomass of different concentrations of TiO2 NPs treated rice. (A) shows root length and shoot height; (B) represents fresh biomass of rice shoots and roots in response to TiO2 NPs exposure. S8
Table S1. Interactions between TiO2 NPs and TC in the hydroponic system TC (mg/l) TiO 2 NPs A1 (500) A2 (1000) A3 (2000) B1 (5) A1B1 A2B1 A3B1 B2 (10) A1B2 A2B2 A3B2 B3 (20) A1B3 A2B3 A3B3 S9
Table S2. Hydrodynamic diameter and zeta potential of TiO2 NPs as affected by TC TC (mg/l) 0 5 10 20 TiO2 NPs (mg/l) Hydrodynamic diameter (nm) Zeta potential (mv) 500 521.4 ± 22.1-15.10 ± 0.97 1000 268.0 ± 10.9-2.75 ± 9.23 2000 64.5 ± 12.7-16.08 ± 5.92 500 235.0 ± 17.7-11.41 ±2.07 1000 144.6 ± 11.3 1.87 ± 11.70 2000 186.5 ± 21.4-1.28 ± 4.59 500 264.5 ± 13.7-10.49 ± 3.65 1000 168.2 ± 25.8 8.73 ± 6.83 2000 65.6 ± 7.2 19.45 ± 6.64 500 325.3 ± 23.5-12.86 ± 1.35 1000 174.1 ± 11.8 1.68 ± 7.67 2000 99.3 ± 16.8 5.71 ± 7.85 S10
Figure S5. Particle distribution of TiO2 NPs in different concentrations of TiO2 NPs amended 1/2x Hoagland s solution as affected by TC. S11
Table S3. Extraction and reaction buffer of antioxidant enzymes Antioxidant enzyme SOD POD Extraction buffer 50 mm phosphate (ph 7.8) containing 0.1% (w/v) ascorbate, 0.1 % (w/v) bovine serum albumin (BSA), and 0.05% (w/v) β- mercaptoethanol 50 mm phosphate (ph 7.0) containing 1% (w/v) polyvinylpyrrolidone CAT 25 mm KH 2PO 4 (ph 7.4) Reaction buffer 100 μl of enzyme extract + 1900 μl of 50 mm phosphate buffer (ph 7.8) containing 9.9 mm L- methionine, 57 μm NBT, 0.0044% (w/v) riboflavin and 0.025% (w/v) Triton X-100 50 μl of enzyme extract + 1.75 ml of 50 mm sodium phosphate buffer (ph 7.0) +0.1 ml of 4% guaiacol in cuvette + 0.1 ml of 1% (v/v) H 2O 2 to initiate the reaction 100 μl of enzyme extract + 1900 μl of 10 mm H 2O 2 Reaction time (min) Wavelength (nm) 20 560 2 470 3 240 S12
Table S4. Methods for joint toxicity evaluation of TC and TiO2 NPs to rice seedlings Method Equation Description Assessment Toxicity Unit (TU) Additional Index (AI) Mixture Toxicity Index (MTI) TUi = Ci/IC50i TU = ΣTUi TU0 = TU/max(TUi) When TU 1, AI = (1/TU)-1; When TU 1, AI = TU(-1)+1 MTI = 1-lgTU/lgTU0 Where TUi is the toxicity unit of the contaminant i; Ci represents the contaminant i caused 50% reduction of fresh biomass in the scenario of single exposure; IC50i represents the concentration of contaminant i that caused 50% reduction of fresh biomass in the scenario of coexposure; TU = 1, additive; TU > TU0, antagonistic; TU < 1, synergistic; TU = TU0, independent; TU0 > TU > 1, partially additive AI > 0, synergistic; AI = 0, additive; AI < 0, antagonistic MTI = 0, independent; 0 < MTI <1, partially additive; MTI = 1, additive; MTI > 1, synergistic; MTI < 0, antagonistic S13
Table S5. Freely dissolved concentration of TC in the co-exposure treatments TC (mg/l) TiO 2 NPs A1 (500) A2 (1000) A3 (2000) B1 (5) 4.013 3.157 1.903 B2 (10) 8.859 7.759 5.736 B3 (20) 18.775 17.561 15.174 Note: The freely dissolved concentration of TC was calculated based on the difference between the addition amount of TC and the portion adsorbed onto TiO2 NPs, which was calculated by the fitting result of Langmuir model. S14
Table S6. Bioaccumulation factor of TC in rice shoots and root Treatment BAF in shoot BAF in root 5 mg/l TC 9.618 119.318 10 mg/l TC 10.397 214.879 20 mg/l TC 10.860 663.690 5 mg/l TC 500 mg/l TiO2 NPs 4.030 4.780 5 mg/l TC 1000 mg/l TiO2 NPs 3.266 2.570 5 mg/l TC 2000 mg/l TiO2 NPs 3.424 1.770 10 mg/l TC 500 mg/l TiO2 NPs 2.289 7.709 10 mg/l TC 1000 mg/l TiO2 NPs 1.485 3.042 10 mg/l TC 2000 mg/l TiO2 NPs 1.524 1.712 20 mg/l TC 500 mg/l TiO2 NPs 2.059 47.667 20 mg/l TC 1000 mg/l TiO2 NPs 2.007 21.302 20 mg/l TC 2000 mg/l TiO2 NPs 0.915 3.377 Note: BAF= CTC in plant tissues/ctc in Hoagland s solution S15
A A B A Figure S6. Interactions between TC and TiO2 NPs in 1/2x Hoagland s solution in the absence of rice. (A) is TC reduction rate at ambient temperature over 5 day; (B) is TC adsorption amount on TiO2 NPs. S16
A B A Figure S7. Phenotypic images of rice seedlings co-exposed to different concentrations of TC and TiO2 NPs in 1/2x Hoagland s solution. (A) shows the images of TC alone and NPs alone treatments; (B) shows the images of co-contaminated treatments. In each image, two seedlings on the left-hand side are the control plants grown in 1/2x Hoagland s solution, and the ones on the right-hand side are the treated plants. S17
Figure S8. The contents of Ti in rice roots treated with different concentrations of TiO2 NPs and TC. S18
Figure S9. Bioaccumulation factor of tetracycline in rice shoots (A) and roots (B) versus freely dissolved concentration of TC in 1/2X Hoagland s solution. S19
A B C Figure S10. The contents of other nutrient elements in rice shoots co-treated with TC and TiO2 NPs. (A) (C) represent the contents of Ca, Mg, and Mo, respectively. S20
Figure S11. The total protein contents in rice shoots (A) and roots (B) upon exposure to TC and TiO2 NPs. S21
A A B A C A D A E A Figure S12. Inhibition rate of rice biomass among all the treatments. (A) and (B) represent biomass inhibition rate in the TC alone and TiO2 NPs alone treatment, respectively; (C) (E) represent biomass inhibition rate in the co-treatments when the TC concentration is at 5, 10, and 20 mg/l, respectively. S22